| Objects: To explore the expression of miRNA-148 b and miRNA-152 in renal cell carcinoma and its function,and further discuss its possible mechanism,which may provide a new idea for early diagnosis and treatment of renal cell carcinoma.Methods:In order to explore the relationship between the expression of miRNA-148 b and miRNA-152 and clinical stage,histological grade of 40 renal cell carcinoma patients,we detected the expression of miRNA-148 b and miRNA-152 in human renal cell carcinoma and paired normal renal tissue through quantitative reverse transcription polymerase chain reaction.Furthermore,miRNA-148 b and miRNA-152 mimics were transmitted into human RCC cell lines 786-O and SN12-PM6,we examined the expression of in different RCC cell lines,and the effects of miRNA-148 b and miRNA-152 on renal cell carcinoma were tested by MTT,cell apoptosis,transwell and colony formation assay.and assessed the impact of miRNA-148 b and miRNA-152 on biological traits such as proliferation,apoptosis,colony formation,invasion and migration of RCC cell at 24 h,48h and 72 h after transfection.Last,bioinformatic search was performed to find target gene of miRNA-148 b and miRNA-152.The expression levels of KIT?AKT?m TOR?Bcl-2?Bcl-2L11 were tested by using Western blot analyses in RCC cell lines 786-O and SN12-PM6 at 24 h,48h and 72 h after transfection with miRNA-148 b and miRNA-152 mimics.Results:Quantitative real time PCR results demonstrated that the relative expression quantity of miRNA-148 b and miRNA-152 was both down-regulated in renal cell carcinoma,and there was a statistically significant difference compared to the paired adjacent normal renal tissues(both P<0.01).and According to the pathological grades,the relative expression of miRNA-148 b and miRNA-152 in well differentiated group(Fuhrman grade I,II)was compared respectively with that in low-moderately differentiated groups(Fuhrman grade ? ? ?),there were both no significant difference(P>0.05).For different clinical stages,compared respectively the relative expression quantity of miRNA-148 b and miRNA-152 in the phage I with that in the phage II-III,it both had no difference between above groups also(P>0.05).In the RCC cell lines 786-O and SN12-PM6,MTT assay showed that inhibitory effect of cell proliferation from miRNA-148 b and miRNA-152 was obvious,there was astatistically significant difference compared to the control group(both P<0.05).Apoptosis were measured after transient transfection of miR-148 b and miR-152,a significant induction of apoptosis could be seen 48 and 72 h after transfection of miR-148 b and miR-152 in the cell line 786-O.The cell line SN12-PM6 was sensitive to miR-148 b and miR-152 resulting in a induction of apoptosis after 48 and 72 h,those of miR-152 reached significant levels after 72 h.For colony formation assay,our results showed that the colony numbers were significantly decreased following miRNA-148 b and miRNA-152 mimics transfection in cell lines 786-O and SN12-PM6,and there was a statistically significant difference between miRNA and negtive control group(both P<0.05).Transwell invasion and migration assay showed that the number of invasion cell after transfection of miRNA-148 b and miRNA-152 across the membrane both were significantly lower than the negative control group in cell lines 786-O and SN12-PM6,with significant difference(both P <0.01).And the similar effect of migration was also observed in cell lines 786-O and SN12-PM6.Biological information search showed that position 1650-1656 bases of KIT3 'UTR can be complementary with seed sequences of miRNA-148 b and miRNA-152.Therefore,we speculate that KIT may be one of target genes of miRNA-148 b and miRNA-152.Western blot analysis proved,in the 786-O cell line,miR-148 b and miR-152 were able to reduce the phosphorylation of KIT and the expression of total KIT 24 h after transfection of miRNAs and adequate controls.It was shown that the phosphorylation of AKT and total AKT was downregulated by miR-148 b and miR-152 in all of the evaluated time points.Looking further downstream of the signaling cascade,miR-148 b and miR-152 almost no effect on p-m TOR as well as on the total m TOR.Bcl-2 expression was downregulated by miR-148 b and miR-152 at all time points.However,the Bcl-2L11 protein expression did not change under miRNA treatment.in the SN12-PM6 cell line,miR-148 b and miR-152 were able to reduce the phosphorylation of KIT at all the evaluated time points.Transfection of the miRNAs showed no effect on KIT protein expression after 24 h.After 48 h and 72 h,a strong reduction of the total KIT expression was observed by transfection of miR-148 b and miR-152.The phosphorylation of AKT and total AKT was reduced by miR-148 b and miR-152 after 24 h,demonstrating a more evident downregulation of AKT expression following transfection of miR-152.The p-m TOR and m TOR western blot analyses in the SN12-PM6 cell line demonstratedalmost no regulation of the protein levels.Bcl-2 expression was reduced in the SN12-PM6 cell line,particularly 48 and 72 h after transfection of miR-148 b and miR-152,but Bcl-2L11 expression was almost unaffected by the transfection of miRNAs.Conlusion:1.The expression level of miR-148 b and miR-152 in human renal cell carcinoma was significantly lower than that in non tumor tissues,but there was no significant correlation with pathological grading and clinical stage of renal cell carcinoma.2.Mi R-148 b and miR-152 have inhibitory effects on the proliferation and apoptosis of 786-O and SN12-PM6 renal cell carcinoma cells,and can inhibit the colony forming ability and invasion and metastasis of 786-O and SN12-PM6 renal cell carcinoma cells.3.KIT is one of the target genes of miR-148 b and miR-152 in renal cell carcinoma,miR-148 b and miR-152 may be through the regulation of KIT,AKT and Bcl-2 expression to inhibit the occurrence and development of renal cell carcinoma. |
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